Alkylcysteine Sulfoxide C–S Monooxygenase Uses a Flavin-Dependent Pummerer Rearrangement

Flavoenzymes are highly versatile and participate in the catalysis of a wide range of reactions, including key reactions in the metabolism of sulfur-containing compounds. S-Alkyl cysteine is formed primarily by the degradation of S-alkyl glutathione generated during electrophile detoxification. A recently discovered S-alkyl cysteine salvage pathway uses two flavoenzymes (CmoO and CmoJ) to dealkylate this metabolite in soil bacteria. CmoO catalyzes a stereospecific sulfoxidation, and CmoJ catalyzes the cleavage of one of the sulfoxide C–S bonds in a new reaction of unknown mechanism. In this paper, we investigate the mechanism of CmoJ. We provide experimental evidence that eliminates carbanion and radical intermediates and conclude that the reaction proceeds via an unprecedented enzyme-mediated modified Pummerer rearrangement. The elucidation of the mechanism of CmoJ adds a new motif to the flavoenzymology of sulfur-containing natural products and demonstrates a new strategy for the enzyme-catalyzed cleavage of C–S bonds.


Materials
All chemicals were purchased from Millipore-Sigma unless specified.LB broth (Lennox formulation) was from EMD Millipore.Kanamycin was from Teknova and IPTG was obtained from Lab Scientific Inc. HPLC and LC-MS solvents were purchased from EMD and were used without further purification.His trap columns (5 ml) were obtained from GE Healthcare.Econo-Pack 10DG and Bio-spin 6 desalting columns were purchased from Bio-Rad Laboratories.Large cultures were grown and overexpressed in 2.5 L baffled ultra-yield flasks from Thomson Instrument Company.NMR tubes (3 mm and 5 mm diameter) were obtained from Wilmad-Labglass.D2O, MeOD, and d6-DMSO were purchased from Cambridge Isotope Laboratories Inc.

Overexpression and purification of enzymes
The genes encoding cmoO and cmoJ were cloned in the pTHT vector (a derivative of the pET28b vector with a TEV protease cleavage site after the N-terminal His-tag).The respective plasmids were transformed into E. coli BL21(DE3) competent cells by electroporation.Starter cultures were grown overnight in LB media containing kanamycin (40 μg/ml).30 ml of this culture were added to 3 L LB media (2 x 1.5 L flasks) with kanamycin (40 μg/ml) and grown at 37 °C with shaking (180 rpm) till OD600~0.6.The flasks were then incubated at 4 °C for ~2 hr without shaking, induced with 500 μM IPTG followed by incubation at 15 °C for ~14 hr with shaking at 180 rpm.The cells were harvested by centrifugation at 5,000 rpm for 20 min and stored in liquid nitrogen until further use.Typical yields were 9-10 g of cell pellet (wet weight) from 3 L cell culture.
For purification, the cell pellets were thawed and resuspended in 40-50 ml of lysis buffer (100 mM KPi, 150 mM NaCl, pH 7.5) at room temperature in the presence of lysozyme (6-8 mg).The suspension was stirred for ~1.5 hr on an ice bath and sonicated to lyse the cells.Cell debris was removed by centrifugation at 15,000 rpm for 20 min and the lysate was filtered using 0.22 µm filters.The filtered lysate was loaded onto a His-trap column pre-equilibrated with lysis buffer.The column was washed with 10 column volumes of wash buffer (100 mM KPi, 20 mM imidazole, 150 mM NaCl, pH 7.5).The protein was then eluted from the His-trap column with elution buffer (100 mM KPi, 250 mM imidazole, 150 mM NaCl, pH 7.5).Protein-containing fractions, identified using the Bradford reagent, were pooled and concentrated using 15 ml 10 kDa filters.The concentrated protein was buffer exchanged to 100 mM KPi, 30% glycerol, pH 7.5 using an Econo-Pac 10DG desalting column.Aliquots of 100 µL desalted enzyme in Eppendorf tubes were flashfrozen in liquid nitrogen and stored at -80 °C until further use.Protein concentration was determined by measuring the absorbance at 280 nm (A280) and utilizing the extinction coefficient calculated using the ProtParam tool of the ExPASy proteomics server (280 = 42400 M -1 cm -1 ).

HPLC conditions:
A. Water B. 100 mM Potassium phosphate buffer, pH 6.6 C. Methanol

LC-MS parameters
LC-ESI-TOF-MS was performed using an Agilent 1260 HPLC system equipped with a binary pump and a 1200 series diode array detector followed by a MicroToF-Q II mass spectrometer (Bruker Daltonics) using an ESI source in negative or positive mode.Analysis was performed on an LC-18-T column (15 cm x 3 mm, 3 μm particles, Supelco).Data were processed using DataAnalysis 4.0 SP1 (Bruker Daltonics).

Utilization of CmoO to synthesize substrates for CmoJ
S-alkyl-N-acetylcysteine was converted to its R-sulfoxide using CmoO.A typical 100 µL reaction contained 200 µM CmoO, 1 µM flavin reductase (FRE), 220 µM FMN, 2-4 mM NADH, and 2 mM S-alkyl-N-acetylcysteine substrate in 100 mM KPi buffer pH 7.5, and was incubated at 37 o C for 1-6 hrs.Protein was then removed using a 10 kDa filter and 40-80 µL of the sample was analyzed by LC-MS.In most cases, substrates were further purified using HPLC and lyophilized.Otherwise, the filtered CmoO reaction mixture was used directly for the CmoJ reaction or lyophilized and later dissolved in anaerobic buffer for anaerobic reactions.

The general formula for determining the percent incorporation of a single stable isotope
The calculation used to determine the percent incorporation of a stable isotope into a reaction product is as follows: 1 If M is the mass of the molecular ion and M' is the mass of the next isotopologue (M' = M+1, M+2, etc.), the contribution of M' in the natural abundance molecule is xA where x is the fractional intensity of M' and A is the peak height (Figure S4A).After the label incorporation, the intensity of M' increases to B1 (Figure S4B), the label incorporation is (B1 -xA1), and the exchangeable pool is (A1 + B1 -xA1).Therefore: .
If y% of the isotope source is labeled this formula becomes:

Anaerobic deuterium incorporation studies on N-acetyl-S-benzylcysteine sulfoxide 3 by CmoJ
For the anaerobic experiments, all buffers and reagents were transferred into an anaerobic chamber (< 5 ppm O2, COY Laboratories).FRE and CmoJ were buffer exchanged into anaerobic 100 mM KPi-D2O pD 7.1, 2 using a Bio-spin 6 desalting column.Stock solutions of substrates (lyophilized solid as mentioned above) and cofactors were also prepared in anaerobic 100 mM KPi-D2O pD 7.1 buffer.A 100 µL reaction mixture, containing 80 µM CmoJ, 1 µM FRE, 100 µM FMN, 1 mM NADH, and 50 µM (single turnover) and 500 µM (multiple turnovers) substrate 3 in 100 mM KPi-D2O buffer pD 7.1, was incubated at 37 o C for 2 hrs.Protein was then removed using a 10 kDa filter, inside the anaerobic chamber and 80 µL of the sample was analyzed by LC-MS.

Deuterium incorporation studies using N-acetyl-S-(cyclopropyl)methylcysteine sulfone 51:
The N-acetyl-S-benzylcysteine sulfone was unsuitable for the exchange study because it showed low levels of non-enzymatic exchange.The study was therefore carried out with N-acetyl-S-(cyclopropyl)methylcysteine sulfone 51 synthesized by chemical over-oxidation of the thioether 50.The enzymatic reactions were performed under both aerobic and anaerobic conditions.FRE and CmoJ were buffer exchanged into 100 mM KPi-D2O pD 7.1, using a Bio-spin 6 desalting column.A 100 µL reaction mixture, containing 80 µM CmoJ, 1 µM FRE, 100 µM FMN, 1 mM NADH, and 500 µM sulfone 51 in 100 mM KPi-D2O buffer pD 7.1, was incubated at 37 o C for 2 hrs.Protein was then removed using a 10 kDa filter, and 80 µL of the sample was analyzed by LC-MS.
For the anaerobic experiments, all buffers and reagents were transferred into an anaerobic chamber (< 5 ppm O2, COY Laboratories).FRE and CmoJ were buffer exchanged into anaerobic 100 mM KPi-D2O pD 7.1, using a Bio-spin 6 desalting column.Stock solutions of substrate 51 and cofactors were also prepared in anaerobic 100 mM KPi-D2O pD 7.1.A 100 µL reaction mixture containing 80 µM CmoJ, 1 µM FRE, 100 µM FMN, 1 mM NADH, and 500 µM sulfone 51 in 100 mM KPi-D2O buffer pD 7.1, was incubated at 37 o C for 2 hrs.Protein was then removed, inside the anaerobic chamber using a 10 kDa filter, and 80 µL of the sample was analyzed by LC-MS (Figure S6) demonstrating that 51 undergoes deuterium exchange under both aerobic and anaerobic conditions.

Kinetics of CmoJ catalyzed reaction with N-acetyl-S-(cyclopropyl)methylcysteine sulfoxide
The assay mixture (100 µL) consisted of 1 µM FRE, 10-50 µM CmoJ, 50 µM FMN, 10-2000 µM substrate 23, and 1 mM 4-mercaptopyridine 3 (55) in 100 mM KPi buffer, pH 7.5.The enzymatic reaction was initiated, at 25 °C, by the addition of 2 mM NADH.The reaction was quenched at suitable time points (30-600 sec) by the addition of 8 M guanidine hydrochloride (100 µL).The enzyme was removed using a 10k kDa cutoff filter and the concentration of product 54 was determined by HPLC analysis by determining the area under the signal at 254 nm.The experiments were performed in duplicates.The assay chemistry is shown in Figure S8A.

Analysis of the CmoJ-catalyzed reaction with N-acetyl-S-(cyclopropyl)methylcysteine for vinyl sulfoxide 26
The reaction mixture consisting of 200 µM CmoJ, 1 µM FRE, 200 µM FMN, 2 mM NADH, 5 mM thiophenol, and S-(cyclopropyl)methyl-N-acetylcysteine sulfoxide 23 solution (final conc.~0.5 mM) in 100 mM KPi buffer pH 7.5 was incubated at 37 o C for 6 hrs.Thiophenol did not inhibit CmoJ or FRE.Protein was removed using a 10 kDa cutoff filter and samples were analyzed using HPLC.An identical reaction mixture (80 L aliquot) was analyzed using LC-MS.No signal corresponding to the mass of 59 ([M+H] + m/z: 344.098) was detected in the EIC.

Synthesis of N-acetyl-S-benzylcysteine 18 O-sulfoxide ([ 18 O]-3) using CmoO
All buffers and reagents were transferred into the anaerobic chamber (< 5 ppm O2, COY Laboratories).FRE and CmoO were buffer exchanged into anaerobic 100 mM KPi pH 7.5, using a Bio-spin 6 desalting column.Stock solutions of substrates and cofactors were also prepared in anaerobic 100 mM KPi buffer, pH 7.5.
A reaction mixture (100 µL, in an Eppendorf tube), consisting of 1 µM FRE, 100 µM CmoO, 100 µM FMN, 4 mM NADH, 2 mM N-acetyl-S-benzylcysteine 2 was made up in anaerobic 100 mM KPi pH 7.5 buffer.The Eppendorf tube was left open and placed in a round bottom flask, which was then capped with a rubber septum and sealed with Teflon tape.The sample was then removed from the anaerobic chamber and purged at least once with a 18 O2-filled balloon.The sample was then incubated with a fresh 18 O2-filled balloon.After 2 hrs, this balloon was replaced with a balloon containing Argon.The round bottom flask was subjected to a low vacuum, to replace most of the oxygen with Argon.The flask was then taken inside the glove box and opened.Protein was filtered using a 10 kDa cutoff filter and 40 µL of the sample was analyzed by LC-MS.The sulfoxide product [ 18 O]-3 was purified by HPLC, lyophilized, and stored at -80 0 C.

Determining the fate of the sulfoxide oxygen during the CmoJ reaction
The synthesized 18 O labeled sulfoxide substrate [ 18 O]-3 was used to determine the fate of the sulfoxide oxygen during the CmoJ-catalyzed reaction.A 100 µL reaction mixture, consisting of 1 µM FRE, 100 µM CmoJ, 100 µM FMN, 2 mM NADH, 1 mM N-acetyl-S-benzylcysteine 18 O-sulfoxide ([ 18 O]-3), and 10 mM PVSu in 100 mM KPi pH 7.5, was incubated at 37 o C for 2 hrs.Protein was removed by ultrafiltration using a 10 kDa cutoff filter and 80 µL of the sample was analyzed by LC-MS.

Determining the source of the sulfenic acid oxygen in the CmoJ product
First, the possibility of molecular oxygen being incorporated into the sulfenic acid was investigated.All buffers and transferred to the anaerobic chamber (< 5 ppm O2, COY Laboratories).FRE and CmoJ were buffer exchanged into anaerobic 100 mM KPi pH 7.5 buffer using a Bio-spin 6 desalting column.Stock solutions of substrates and cofactors were also prepared in anaerobic 100 mM KPi buffer, pH 7.5 buffer.
A reaction mixture (100 µL, in an Eppendorf tube) consisting of 1 µM FRE, 100 µM CmoJ, 100 µM FMN, 2 mM NADH, 1 mM N-acetyl-S-benzylcysteine sulfoxide 3, and 10 mM PVSu was made in anaerobic 100 mM KPi pH 7.5 buffer.The Eppendorf tube was left open and placed in a round bottom flask, which was then capped with a rubber septum and sealed with Teflon tape.The sample was then removed from the anaerobic chamber and purged at least once with a 18 O2filled balloon.The sample was then incubated with a fresh 18 O2-filled balloon.After 2 hrs, this balloon was replaced with a balloon containing Argon.The round bottom flask was subjected to a low vacuum, to replace most of the oxygen with Argon.The flask was then opened inside the glove box.Protein was removed by ultrafiltration using a 10 kDa cutoff filter and 80 µL of the sample was analyzed by LC-MS.
N-acetyl-S-(4,5-dimethoxy-2-nitrobenzyl)-L-cysteine sulfoxide 42: In a 10 mL round-bottomed flask, N-acetyl-S-(4,5-dimethoxy-2-nitrobenzyl)-L-cysteine (64, 15 mg, 0.04 mmol) was dissolved in 2 mL acetic acid.The reaction was cooled to 10 o C and H2O2 (15 L of 33% w/v) was added.The reaction was stirred at 10 o C for 15 mins and then acetic acid was removed at 0 o C (avoid heating up to reduce the risk of overoxidation) by a stream of air.The residue was washed with diethyl ether (2 x 10 mL) to obtain a light brown solid.This method of making the sulfoxide produces a mixture of diastereomers, as the reaction is not stereoselective. 1

Photo-generation of N-acetylcysteine sulfenic acid and its in situ trapping with phenyl vinyl sulfone
For the photochemical reactions, a uvBeast™ 365 nm LED light was used.A typical reaction mixture contained 1 mM sulfoxide 42 in 100 mM KPi pH 7.5.The reaction was carried out in an Eppendorf tube that was covered with aluminum foil with the reflective surface on the inside.The cap of the tube was left open, and the light was set up from the top, to focus the beam on the lower tip of the Eppendorf tube.The solution was illuminated for 2 hrs.at 37 o C and analyzed for substrate consumption by HPLC.
To trap the sulfenic acid product, PVSu (40) was used at the same concentration used to trap the sulfenic acid in the enzymatic reaction.A typical reaction mixture contained 1 mM of sulfoxide 42 and 10 mM PVSu in 100 mM KPi pH 7.5.The solution was illuminated for 2 hrs.at 37 o C as described above and analyzed for the trapped product 41 by LC-MS.
An identical reaction carried out in 70% H2 18 O-KPi buffer (100 mM pH ~7.5) with 1 mM sulfoxide 42 and 10 mM PVSu did not show any incorporation of 18 O into the trapped sulfenic acid 41.However, lowering the concentration of PVSu to 2 mM, while keeping the substrate 42 at 1 mM resulted in ~60% incorporation of the available 18 O into the trapped sulfenic acid.a.The level of incorporation was calculated based on the formula derived from Figure S4.

Evaluation of sulfenic acid oxygen exchange during the CmoJ-catalyzed reaction
To determine the origin of the sulfenic acid oxygen in the CmoJ product, it was necessary to compare the extent of oxygen exchange, in sulfenic acid generated under similar conditions, in the enzymatic and photochemical systems.This was accomplished by running reactions in which PVSu was in excess (10 mM), substrate concentrations [3 (enzymatic)] = [42 (photochemical)] = 1 mM, and by matching the light intensity and the enzyme concentrations to equalize the rates of cysteine sulfenic acid production.
Photochemical generation of the sulfenic acid 4: A typical reaction mixture contained 1 mM of sulfoxide 42 in 100 mM KPi pH 7.5 in a foil-covered Eppendorf tube as described above.The reaction was illuminated through a 0.3 OD filter (Edmund Optics stock #46-212, transmittance = 0.5).Samples were collected every 5 mins and analyzed for substrate consumption by HPLC (Figure S18).
To determine the amount of solvent oxygen incorporation in PVSu-trapped sulfenic acid, a typical photochemical reaction contained 1 mM of sulfoxide 42 and 10 mM PVSu in 50% H2 18 O-100 mM KPi pH 7.5 in a foil-shielded Eppendorf tube and illuminated through the 0.3 OD filter.For the corresponding enzymatic reaction, a reaction mixture containing 100 µM CmoJ, 1 µM FRE, 100 µM FMN, 4 mM NADH, 10 mM PVSu, and 1 mM N-acetyl-S-benzyl cysteine sulfoxide 3 in 50% H2 18 O-KPi 100 mM pH 7.5 was incubated at 25 0 C. Samples were collected every 5 minutes from each of the reactions and analyzed by LC-MS (Figure S18).

Figure S4 :
Figure S4: Calculation of percent isotope incorporation of stable isotopes into small molecules, using LC-MS data of natural abundance and labeled species.A) A typical LC-MS spectrum showing the natural isotopic distribution of a molecule.B) An LC-MS spectrum after isotope labeling showing the enhanced M' signal.

Figure S5 :
Figure S5: Lack of deuterium incorporation from solvent into N-acetyl-S-benzylcysteine sulfoxide by CmoJ in the absence of oxygen.A) Proposed deuterium incorporation into N-acetyl-S-benzylcysteine sulfoxide by CmoJ in the absence of oxygen, B) LC-MS analysis of the reaction mixture resulting from anaerobic incubation of CmoJ + N-acetyl-S-benzylcysteine sulfoxide in D2O (left panel) and H2O (right panel).There is no apparent increase in the m/z = 271 peak on incubation with D2O.

Figure S6 :
Figure S6: CmoJ-catalyzed incorporation of deuterium into N-acetyl-S-(cyclopropyl)methylcysteine sulfone 51.A) Proposed CmoJ-catalyzed deuterium incorporation into 51.B) CmoJ full reaction with 51 under aerobic conditions.C) Reaction with 51 in the absence of CmoJ under aerobic conditions.D) CmoJ reaction in the absence of FMN under aerobic conditions.E) CmoJ full reaction with 51 under anaerobic conditions.F) Reaction with 51 in the absence of CmoJ under anaerobic conditions.G) CmoJ reaction in the absence of FMN under anaerobic conditions.The full reaction, under aerobic and anaerobic conditions, exhibits ~17% incorporation of 2 H (Figure S4).

Figure S10 :
Figure S10: Strategy to detect vinyl sulfoxide 26 using thiophenol trapping.A) Proposed formation of 26 in the CmoJ-catalyzed reaction of N-acetyl-S-(cyclopropyl)methylcysteine.B) HPLC chromatogram (254 nm) of the CmoJ reaction run in the presence of thiophenol.No new peak was observed in the full reaction (Blue: full reaction + thiophenol, red: no CmoJ + thiophenol, green: no substrate + thiophenol).Inset is the EIC for 59 ([M+H] + m/z = 344.09) in the reaction mixture, and shows that 59 was not formed at detectable levels.

Figure S14 :Figure S15 :
Figure S14: Determination of the origin of the sulfenic acid oxygen.A) LC-MS analysis of 41 derived from 18 O-labeled sulfoxide ([ 18 O]-3)showing that the sulfenic acid oxygen is not derived from the sulfoxide.B) LC-MS analysis of 41 derived from unlabeled sulfoxide and 18 O2 showing that the sulfenic acid oxygen is not derived from 18 O2.C) LC-MS analysis of the CmoJ reaction with 3 run in H216  O buffer and D) CmoJ reaction run in ~70% H2 18 O buffer; demonstrating that the sulfenic acid oxygen is buffer derived (~ 87% incorporation of available 18 O).

Figure S18 :
Figure S18: Incorporation of oxygen from water into the photochemically and enzymatically produced sulfenic acid 4. A) LC-MS of the trapped photogenerated sulfenic acid 41, after 50% conversion, showing no incorporation of 18 O; B) LC-MS of the enzymatically generated sulfenic acid 41, after 50% conversion, showing ~80% incorporation of available 18 O; C) A plot of sulfenic acid formed enzymatically (blue circles) and photochemically (orange circles) from 1 mM substrate, D) A plot of the amount of 18 O incorporated into sulfenic acid formed enzymatically (blue circles) and photochemically (orange circles) from 1 mM substrate 3 (enzymatic) and 42 (photochemical).

Figure S19 :
Figure S19: Trapping FMN N5-peroxide intermediate 46 6 in the CmoJ-catalyzed reaction.A) Trapping of the FMN N5-peroxide intermediate, as a stable FMN N5-oxide, when the CmoJ reaction is carried out in the presence of an inactive substrate analog 2, B) LC-MS analysis showing the formation of FMN N5 oxide 46 only in the presence of photo-reduced FMN, substrate analog 2, and CmoJ (EIC of m/z = 473 [M+H] + , Green: full reaction, brown: no substrate analog, blue: no enzyme).Insert shows the MS of the FMN N5 oxide formed in the reaction mixture.

Table S1 :
Effect of trapping agent concentration on the exchange of N-acetylcysteine sulfenic acid 4 with buffer.a